SUMMARY OF WORK: Our lab is focused on understanding the role of the ShcC adaptor protein in RTK function in general and neural development in particular. We have shown through a combination of techniques that ShcC expression is specific to differentiating neurons. Based on these results, we examined ShcC expression in a variety of human, mouse and rat cell lines. This analysis indicated that ShcC expression is limited to human neuroblastoma tumor cell lines further supporting the notion that ShcC functions in neurons. Furthermore, these results suggested a potential role for ShcC in neuroblastoma (NB) tumor formation. IN contrast, we did not detect ShcC expression in other nervous system tumors such as peripheral neuroepithelioma tumors (PNETs). In collaboration with Bernard Weissman's laboratory, we have examined the expression of the ShcC in cell hybrids between human NB and PNET tumor cell lines. The resulting hybrid cell lines retain the PNET phenotype as assessed by expression of various markers of differentiation. Interestingly, the cell hybrids have suppressed ShcC expression normally found in NB cells. These findings further support the notion that ShcC expression is specific to NB tumors. Furthermore, this system provides a useful model for understanding the regulation of ShcC expression at the transcriptional level. We are currently testing whether forced expression of ShcC (using ShcC retroviral expression vectors) in the cell hybrids might shift the resulting ShcC expressing cells to a more NB phenotype. To examine the importance of ShcC for RTK function, we have tested if mutant ShcC proteins block RTK signaling. Although Shc proteins are implicated in the activation of the Ras-MAPK signal transduction pathway by virtue of their ability to couple to the Grb2-Sos signaling complex, their importance in RTK activation of Ras is unclear. Expression of the isolated ShcC SH2 domain results in potent inhibition of EGFR activation of MAPK in the absence of stable association with the receptor. In contrast, the isolated PTB domain complexes with the EGFR and inhibits binding of full length Shc to the receptor yet does not significantly inhibit EGFR activationof MAPK. These results suggest that the SH2 domain forms a complex with another cellular protein which may be required for EGFR function. We are currently in the process of identifying this protein or proteins using a combination of approaches, including yeast-two hybrid analysis and bacterial expression cloning. Furthermore, given the efficacy of these ShcC dominant-negative proteins, we are using these mutant proteins to assess the importance of ShcC in neuronal development and signaling.